硫氧还蛋白-(His)_ 6 融合的瑞替普酶的表达、纯化和活性检测(英文)  

A Thioredoxin-(His)_6 Fusion Protein of Reteplase:Expression,Purification,and Activity Analysis

作　　者：张新元[1] 廖建民[1] 孙石静[1] 沈子龙[1] 

机构地区：[1]中国药科大学生物制药学院,南京210009

出　　处：《中国药科大学学报》2004年第4期374-378,共5页Journal of China Pharmaceutical University

基　　金：江苏省科技厅基金资助项目 (No .BG2 0 0 2 318)~~

摘　　要：目的 :构建硫氧还蛋白 (His) 6 瑞替普酶融合表达载体 ,提高瑞替普酶在大肠杆菌中的表达量 ,简化rPA的分离纯化。方法 :将rPA基因克隆于原核表达载体pET32a硫氧还蛋白 (组氨酸 ) 6标签下游 ,在大肠杆菌BL2 1(DE3)中用乳糖进行诱导表达。采用一步稀释法对融合蛋白体外复性后 ,使用Ni2 + 亲和层析柱 ,对包涵体复性液进行初步纯化。采用体外溶圈法对复性后和纯化后的融合蛋白进行生物活性的测定。结果 :得到分子量约为 5 6kDa的融合蛋白 ,表达量达到总蛋白的 30 %以上。比本实验室构建的非融合表达载体表达量提高了约 5 0 %。经过一步Ni2 + 亲和层析 ,融合蛋白纯度达到80 %以上。体外溶圈法实验表明融合蛋白复性后和纯化后具有溶栓活性。结论 :与非融合表达相比 ,融合表达的表达量明显提高。即使N端额外融合一段融合多肽 ,rPA仍然具有生物学活性。AIM:To increase the expression amount of reteplase,a vector was constructed which expressed the fusion protein thioredoxin-(His) 6-rPA. The purification of reteplase was simplified by one-step affinity chromatography in this study. METHOD: Reteplase gene was inserted into the prokaryotic expression vector pET32a and was fused to the downstream of the thioredoxin and the (His) 6Tag in the same reading frame. The fusion protein was expressed in E. coli BL21 (DE3) induced by lactose. After refolded in vitro by one-step dilution,the fusion protein was simply purified using Ni 2+-chelating chromatography. Fibrin plate assay was used to detect the bioactivity of the fusion protein. RESULT:A fusion protein of 56 kD was obtained. The amount of the fusion protein was more than 60% of the total bacterial protein. Comparing with the non-fusion reteplase,the yields of target protein increased about 50%. The purity of the fusion protein reached above 80% after one-step purification using Ni 2+-chelating chromatography. The bioactivity of the refolded and purified fusion protein was detected. CONCLUSION: Expression level of the fusion protein increased distinctively compared with that of the non-fusion protein. Bioactivity of reteplase was detected even though a fusion peptide was fused to the N-terminal of reteplase.

关 键 词：硫氧还蛋白 (组氨酸)6标签 瑞替普酶 融合蛋白 表达 纯化 活性 

分 类 号：TQ464[化学工程—制药化工]